Crystalline pyridazine compound

ABSTRACT

A crystalline compound of formula (1) and its salts and solvates are provided for the treatment or prophylaxis of hepatitis C virus infections 
     
       
         
         
             
             
         
       
     
     Methods of making and formulating crystalline compound (1) are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application Ser.No. 60/958,595 entitled “Crystalline Pyridazine Compound and UseThereof,” filed Jul. 6, 2007. The content of this provisionalapplication is herein incorporated by reference in its entirely for allpurposes.

BACKGROUND OF THE INVENTION

The hepatitis C virus is an enveloped, single-stranded, positive senseRNA virus in the family Flaviviridae. HCV mainly replicates withinhepatocytes in the liver. Circulating HCV particles bind to receptors onthe surfaces of hepatocytes and subsequently enter the cells. Onceinside the hepatocyte, HCV utilizes the intracellular machinerynecessary to accomplish its own replication. Lindenbach, B. Nature436(7053):932-8 (2005). The HCV genome is translated to produce a singleprotein of around 3011 amino acids. This “polyprotein” is thenproteolytically processed by viral and cellular proteases to producethree structural (virion-associated) and seven nonstructural (NS)proteins.

HCV encodes two proteases, the NS2 cysteine autoprotease and the NS3-4Aserine protease. The NS proteins then recruit the viral genome into anRNA replication complex, which is associated with rearranged cytoplasmicmembranes. RNA replication takes places via the viral RNA-dependent RNApolymerase of NS5B, which produces a negative-strand RNA intermediate.The negative strand RNA then serves as a template for the production ofnew positive-strand viral genomes. Nascent genomes can then betranslated, further replicated, or packaged within new virus particles.New virus particles presumably bud into the secretory pathway and arereleased at the cell surface.

HCV has a high rate of replication with approximately one trillionparticles produced each day in an infected individual. Due to lack ofproofreading by the HCV RNA polymerase, HCV also has an exceptionallyhigh mutation rate, a factor that may help it elude the host's immuneresponse.

Based on genetic differences between HCV isolates, the hepatitis C virusspecies is classified into six genotypes (1-6) with several subtypeswithin each genotype. Subtypes are further broken down into quasispeciesbased on their genetic diversity. The preponderance and distribution ofHCV genotypes varies globally. For example, in North America genotype 1apredominates followed by 1, 2a, 2b, and 3a. In Europe genotype 1 ispredominant followed by 2a, 2b, 2c, and 3a. Genotypes 4 and 5 are foundalmost exclusively in Africa. Genotype is clinically important indetermining potential response to interferon-based therapy and therequired duration of such therapy. Genotypes 1 and 4 are less responsiveto interferon-based treatment than are the other genotypes (2, 3, 5 and6). Duration of standard interferon-based therapy for genotypes 1 and 4is 48 weeks, whereas treatment for genotypes 2 and 3 is completed in 24weeks.

The World Health Organization estimates that world-wide 170-200 millionpeople (3% of the world's population) are chronically infected with HCV.Approximately 75% of these individuals are chronically infected withdetectable HCV RNA in their plasma. These chronic carriers are at riskof developing cirrhosis and/or liver cancer. In studies with a 7-16years follow-up, 7-16% of the patients developed cirrhosis, 0.7-1.3%developed hepatocellular carcinoma and 1.3-3.7% died of liver-relateddisease.

The only treatment option available today is the use of interferon α-2(or its pegylated form) either alone or combined with ribavirin.However, sustained response is only observed in about 40% of thepatients and treatment is associated with serious adverse effects. Thereis thus an urgent need for potent and selective inhibitors of HCV.

Relevant disclosures include U.S. Pat. Nos. 4,914,108; 4,988,707;4,990,518; 5,137,896; 5,208,242; 5,227,384; 5,302,601; 5,374,638;5,405,964; 5,438,063; 5,486,525; 6,479,508; and U.S. Patent PublicationNo. US2003/0108862 A1, Canadian Patent No. 2423800 A1, German PatentNos. 4211474 A1, 4236026, 4309969, 4318813, European Patent Nos. EP 0138 552 A2, EP 0 706 795 A2, EP 1 132 381 A1, Great Britain Patent No.2158440 A, PCT Patent Publication Nos. WO 00/20416, WO 00/39127, WO00/40583, WO 03/007945 A1, WO 03/010140 A2, WO 03/010141 A2, WO93/02080, WO 93/14072, WO 96/11192, WO 96/12703, WO 99/27929,PCT-US2004/43112, PCT-BE2003/000117, PCT-US2005/26606, Akamatsu, et al.,“New Efficient Route for Solid-Phase Synthesis of BenzimidazoleDerivatives”, 4:475-483, J. COMB. CHEM., 2002, Baginski S G et al.,Proc. Natl. Acad. Sci. U.S.A. 2000 Jul. 5; 97(14):7981-6). Cleve et al.,“Derivate des Imidazo[4.5-b]—und Imidazo[4.5-c]pyridins”, 747:158-171,JUSTUS LIEBIGS ANNALEN DER CHEMICA, 1971, Kiyama, et al., “Synthesis andEvaluation of Novel Nonpeptide Angiotensin II Receptor Antagonists:Imidazo[4,5-c]pyridine Derivatives with an Aromatic Substituent”,43(3):450-60, CHEM PHARM BULL, 1995, Mederski et al., “Synthesis andStructural Assignment of Some N-substituted ImidazopyridineDerivatives”, 48(48):10549-58, TETRAHEDRON, 1992, Yutilov et al.,23(1):56-9, KHIMIKO-FARMATSEVTICHESKII ZHURNAL, 1989. In addition, seeWO 05/063744.

The compound of formula (1) is the subject of WO 08/005,519.

Compound 1) as produced by the process of WO 05/063744 is substantiallyor entirely amorphous. It is believed to be a hydrate (hereafter“amorphous” compound (1)).

It is an object of this invention to provide compound (1) in crystallineform.

SUMMARY OF THE INVENTION

In accordance with achieving the foregoing objects of this invention, acrystalline compound is provided having formula (1)

and its salts, which is substantially free of amorphous compound (1).

In an embodiment, the crystalline compound (1) is the free basesubstantially free of amorphous compound (1) and any other crystal formof compound (1).

Another embodiment of this invention is a method for making crystallinecompound (1)

comprising crystallizing compound (1) from crystallization solvent andcontrolling the amount of water in the crystallization solvent.

In another embodiment, a composition is provided that comprisescrystalline free base compound (1) which is substantially free of thechloride salt of compound (1).

Crystalline compound (1) is useful in a method for therapy orprophylaxis of HCV infection comprising administering to a subject atherapeutic or prophylactic dose of crystalline compound (1). Anotherembodiment comprises the use of crystalline compound (1) for themanufacture of a medicament for the prevention or treatment of an HCVinfection in a mammal (more specifically a human).

Another embodiment of this invention relates to pharmaceuticalcompositions of the crystalline formula (1) compound comprising at leastone pharmaceutically acceptable excipient. In one embodiment thecompound of formula (1) is formulated with an organic acid andoptionally formulated into a pharmaceutical dosage form such as acapsule. In another embodiment, crystalline compound (1) is micronizedand formulated as a suspension.

Crystalline compound (1) or the pharmaceutical compositions of thisinvention are employed in the treatment or prophylaxis of hepatitis C.

Crystalline compound (1) exhibits improvements in pharmacologicalfeatures and cost advantages, in particular improved purity, storagestability and manufacturing reproducibility. A particular advantage isits higher melting point as compared to the amorphous form.

Other features of this invention, including novel intermediates andproduct compositions, will be apparent from consideration of thisapplication as a whole.

FIGURES

FIG. 1 depicts an X-ray powder diffraction (XRPD) pattern obtained forcrystalline compound (1) reference standard obtained by the method ofexample 1.

FIG. 2 depicts another X-ray powder diffraction pattern obtained forcrystalline compound (1).

FIG. 3 is an X-ray powder diffraction pattern obtained for the amorphousform of compound (1) Research Lot 6, obtained by the method of Example1a in WO 08/005,519.

FIG. 4 illustrates a DSC thermogram obtained for crystalline compound(1) reference standard, 1° C./min scan, obtained by the method ofExample 1 below.

FIG. 5 shows a DSC thermogram obtained for the amorphous form ofcompound (1) Research Lot 6, 5° C./min scan, obtained by the method ofExample 1a in WO 08/005,519.

DETAILED DESCRIPTION OF THE INVENTION

Crystalline compound (1) is defined as a solid comprising compound (1)in which the constituent molecules are packed in a regularly orderedrepeating pattern extending in all three spatial dimensions.Identification of crystallinity is readily accomplished in a number ofways known to those skilled in the art. Microscopic examination of thetest composition often will reveal the presence of regular shapes,suggesting ordered internal structure. In the case of the crystalembodiment produced in example 1, the regular shape generally is rod orneedle-like.

XRPD is another method for identifying crystalline compound (1). Theregularly ordered structure of constituent molecules in a crystaldiffracts incident x-rays in a distinct pattern depicted as a spectrumof peaks. This pattern of peaks for crystalline compound (1) is shown inFIGS. 1 and 2. On the other hand, FIG. 3 depicts an XRPD forsubstantially amorphous compound (1), which lacks distinct peaks. Whilethe XRPD peaks for crystalline compound (1) may vary in intensity thesame general pattern will be present in replicate x-ray diffractionanalysis.

Crystalline compound (1) exhibits an XRPD dominant peak(s) at about 17degrees theta 20, ordinarily 17.4 and 17.5. By “about” applicants meanwithin the typical variation in measurement of XRDP peaks. Suchvariations may result from the use of different instruments, instrumentsettings, batches of product, post-crystallization processing such asmicronization or milling, and with varying sample preparation methods.In general, “about” means+0.5 degree theta 20. An example of this sortof variation can be seen by comparing FIGS. 1 and 2. In particular, peakintensity (e.g., at about 30) may vary due to crystal orientationeffects.

Illustrative examples of other dominant peaks for crystalline compound(1) are at about 8, 10, 13, 16, 19 and 24 degrees theta 20, ordinarily8.4, 10.0, 13.5, 15.7, 16.8, 16.9, 18.8 and 24.4. Any one or more ofthese peaks (but especially, 8, 10, 15.7, 16.7 and 16.9, with or withoutthe peaks at about 17, are suitably employed to define the XRDP forcrystalline compound (1).

The identification of a crystal form of compound (1) need not requirethe presence of any one or more of the dominant peaks seen in FIG. 1 or2. Rather, the presence or absence of dominant peaks ordinarily is takeninto account with other diagnostic characteristics (e.g., DSCthermogram) to identify a candidate as crystalline compound (1).

Crystalline compound (1) also is characterized by DSC thermogram, whichreveals an endothermic onset at about 235° C. in differential scanningcalorimetry profile. Typically, some variation in this measurement alsowill be encountered (usually, ±1-3° C.).

Crystalline compound (1) also is characterized by its heat of fusion(DH_(f)) of about 81 J/g (42 KJ/mole).

Crystalline compound (1) is made by a process comprising dissolvingcompound (1) in solvent and forming crystals therefrom. Typical solventsfor use herein are ethyl acetate, isopropyl alcohol or a cosolventcontaining ethyl acetate and isopropyl alcohol. Other suitable solventsare obtained from the solubility map in McConville, F.X. “Pilot PlantReal Book” (2002) which plots the dielectric constant and Hildebrandsolubility parameter for a variety of solvents.

Solvents close to ethyl alcohol and isopropyl alcohol on the map(dielectric 2.5-20 and Hildebrand 15-24) are ethyl ether, isobutylacetate, butyl acetate, anisole, chlorobenzene, chloroform, methylacetate, THF, dichloromethane, dichloroethane, 1,2-dichlorobenzeke,methylisobutylketone, methylethylketone, cyclohexanone, acetone,1-butanol, 2-methoxyethanol, isobutanol, 2-butanol, cyclohexanol,isoamyl alcohol, pyridine, methyl formate, 1-pentanol, and/or2-butoxyethanol.

Some of these solvents would not be preferred due to toxicity issues,but this could be overcome by careful solvent removal from the product.It will be within the skill of the ordinary artisan to conductlaboratory screening to determine suitability of a candidate solvent forthe preparation of crystalline compound (1). Combinations of thesesolvents also fall within the scope of the invention.

A key finding facilitating the preparation of crystalline compound (1)is that the water content of the crystallization solvent must becontrolled in order to obtain and/or optimize the production ofcrystalline product. For example, when using ethyl acetate as solvent,the upper limit on water content is about 0.6% to 0.9% by weight.

An additional consideration with regard to water content is its use toremove other forms of compound (1) that are less soluble than thecrystalline free base in liquid lipophilic pharmaceutical carriers. Forexample the chloride salt of compound (1) is less soluble than the freebase in the fatty acid solutions employed as carriers herein. Insufficiently large amounts such salts produce an undesirable haze in thepharmaceutical product. The final synthetic step of example 1 produces amixture of free base together with minor amounts of the chloride salt.The haze-producing chloride salt is removed by first dissolving theproduct in a solvent containing a relatively high amount of water (about3%-10%) at alkaline pH. Refluxing in this solvent assures that there isenough water to back convert the chloride salt to the free base.Thereafter, the crystalline free base is crystallized from this solvent.This process optionally is repeated with decreasing water concentrationsto gradually remove the chloride salt from the product. The final stepis then accomplished with low water content (usually less than about0.9% water) in order to crystallize the free base substantially free ofthe amorphous compound (1). In general, haze in the pharmaceuticalpreparation is not encountered when the chloride content in the finalproduct is ordinarily less than about 100 ppm. The amounts of wateremployed will vary depending upon the concentration of contaminatingchloride salt and other experimental variables determinable by theskilled artisan. In summary, the water content of the crystallizationsolvent is controlled, both to convert chloride (or other relativelywater soluble salts of compound (1)) and to avoid generation ofamorphous compound (1).

The amount of permitted water for each function will vary depending uponthe solvent or solvents employed for crystallization, the concentrationof compound (1), the temperature of the crystallization step, the timeof crystallization, the tolerable amount of amorphous compound (1), andother variables. Hence, it will be incumbent upon the artisan todetermine the optimal water level for obtaining the desired results,usually by conducting a typical variable matrix study. The lowest waterconcentration for avoiding generation of the amorphous compound (1) ismore a matter of practical economics. For example, 0.05% water by weightis acceptable.

In general, the final crystallization step is conducted in substantiallyanhydrous solvent. Substantially anhydrous solvent is defined as solventcontaining a sufficiently small amount of water that the resultingproduct contains crystalline compound (1) and is substantially free ofamorphous compound (1), typically less than about 40%, ordinarily lessthan about 30, 20, 10, 5, 3, 2 or 1% by weight of amorphous compound (1)in the total of all forms of compound (1) in the product composition.

In general, substantially anhydrous solvent will about 0.5%-0.9% waterby weight of the crystallization solvent. However, more water can bepresent if the desired product is permitted to contain the greaterproportions of amorphous compound (1). However, it is optimal if thecompound (1) composition is free of detectable amorphous compound (1).

The water content is controlled by any manner results in the properamount of water in the crystallization step concerned. When formation ofamorphous compound (1) is to be avoided, suitable techniques forminimizing or reducing the amount of water include adding drying agentsand/or azeotropically removing water. It is most convenient to removewater during reflux dissolution of compound (1) just prior tocrystallization. Of course, control of water content includes addingwater as well, as will typically be the case during steps to convert thechloride salt.

Amorphous compound (1) optionally is used as starting material forcrystallization (form conversion). Alternatively, crystallization isconducted directly from the final reaction products without anintermediate recovery of amorphous compound (1). The crystallizationtypically is conducted by providing or dissolving compound (1) insolvent or solvent mixture at reflux (sufficient to dissolve compound(1), about 1 to 5 hours), followed by cooling to about 18-23° C. over4-8 hours, then optionally agitated for about 8 to 20 hours at about18-23° C. Agitation is optional but increases the rate ofcrystallization. Reflux is not critical since all that is necessary isthat compound (1) be placed in solution. However, refluxing compound (1)has the advantage of rapidly dissolving compound (1) and azeotropicallyremoving water at the same time. Water is controlled beforecrystallization starts or during crystallization, or both, although ingeneral it is best to reduce water below the desired limit before anycompound (1) can precipitate as the amorphous polymorph.

Generation of amorphous material is optimized by using relatively longercrystallization times, higher temperatures and lower concentrations ofcompound (1). Determining the various optimal crystallization processparameters are well within the skill of the ordinary artisan.

An embodiment herein is a composition made by the process of combiningcrystalline compound (1) with a pharmaceutically acceptable excipientand forming a pharmaceutical dosage form such as a tablet or capsule.The resulting product need not contain crystalline compound (1). Whileit is expected that dosage forms made from crystalline compound (1) willcontain only compound (1) in crystalline form. However, in someembodiments the crystalline compound (1) is an intermediate fordissolution in the carrier or excipient.

The crystalline compound (1) of this invention is administered to asubject mammal (including a human) by any means well known in the art,i.e. orally, intranasally, subcutaneously, intramuscularly,intradermally, intravenously, intra-arterially, parenterally or bycatheterization in a therapeutically effective amount, i.e., anHCV-inhibiting amount or an HCV-replication inhibiting amount. Thisamount is believed to be an amount that ensures a plasma level of about100 nM, 3 times the protein-adjusted EC90. This ordinarily is expectedto be achieved by daily oral administration of about 0.5-about 5 mg/kg,typically about 0.7 to 2.2 mg/kg, most ordinarily about 1.2 mg/kgbodyweight for humans.

The optimal dosage of the compound of this invention will depend uponmany factors known to the artisan, including bioavailability of thecompound in a given formulation, the metabolism and distribution of thecompound in the subject, the fasted or fed state of the subject,selection of carriers and excipients in the formulation, and otherfactors. Proper dosing typically is determined in the preclinical andclinical settings, and is well within the skill of the ordinary artisan.The therapeutically effective amount of the compound of this inventionoptionally is divided into several sub-units per day or is administereddaily or in more than one day intervals, depending upon the nature ofthe infection, the patient's general condition and the formulation ofthe compound of this invention. Generally, the compound is administeredtwice daily.

The compound of this invention is employed in concert with other agentseffective against HCV infections. They optionally are administeredseparately in a course of therapy, or are combined with compound (1) ina unitary dosage form such as tablet, iv solution or capsule. Such otheragents include, for instance, interferon-alpha, ribavirin, and/orcompounds falling within the disclosures of EP1162196, WO 03/010141, WO03/007945, WO 00/204425 and/or WO 03/010140 (and other filings withintheir patent families). Other agents for administration in a course oftherapy with the compound of this invention include compounds now inclinical trials, in particular HCV protease inhibitors such as VX-950(Vertex Pharmaceuticals), SCH 5030347 (Schering Plough) and BILN-2061(Boehringer Ingelheim), nucleoside HCV inhibitors such as NM283, NM107(both Idenix/Novartis) and R1626 (Hoffmann-LaRoche), and non-nucleosideHCV inhibitors including HCV-086 and -796 (both ViroPharma/Wyeth).Supplementary antiviral agents are used in conventional amounts. If theefficacy of the compound of this invention and the supplementarycompound are additive then the amounts of each active agent optionallyare commensurately reduced, and more so if the agents actsynergistically. In general, however, the agents are used in theirordinary active amounts in unitary combination compositions.

Co-administered agents generally are formulated into unitarycompositions with the compound of this invention so long as they arechemically compatible and are intended to be administered by the sameroute. If not, then they optionally are provided in the form of amedical kit or package containing the two agents in separaterepositories or compartments.

The compound of this invention typically is provided as the free base,but also optionally is prepared as a salt. Salts typically are preparedby acid addition of organic and/or inorganic acids to the free base.Examples include (1) inorganic acids such as hydrohalogen acids, e.g.hydrochloric or hydrobromic acid, sulfuric acid, nitric acid, phosphoricacid and sulfamic acids; or (2) organic acids such as acetic, propanoic,hydroxyacetic, benzoic, 2-hydroxypropanoic, 2-oxopropanoic, lactic,fumaric, tartaric, pyruvic, maleic, malonic, malic, salicylic (e.g.2-hydroxybenzoic), p-aminosalicylic, isethionic, lactobionic, succinic,oxalic and citric acids; organic sulfonic acids, such asmethanesulfonic, ethanesulfonic, benzenesulfonic, p-toluenesulfonic,C1-C6 alkylsulfonic, benzenesulfonic, p-toluenesulfonic, andcyclohexanesulfamic acids. Typical salts are the chloride, sulfate,bisulfate, mesylate, besylate, esylate, phosphate, oxalate, maleate,succinate, citrate, malonate, and/or fumarate salts. Also includedwithin the scope of this invention are the salts of the compound of thisinvention with one or more amino acids, typically naturally-occurringamino acids such as one of the amino acids found in proteins. The acidiccounterion desirably is physiologically innocuous and non-toxic orotherwise pharmaceutically acceptable, unless the salt is being used asan intermediate in preparation of the compounds whereupon toxicity isnot relevant. Ordinarily, compound (1) will be administered as the freebase, but suitable salts include mesylate (methanesulfonic acid) andHCl.

The compound of this invention includes the solvates formed with thecompound of this invention or their salts, such as for example hydrates,alcoholates and the like.

The pharmaceutical compound of this invention optionally is formulatedwith conventional pharmaceutical carriers and excipients, which will beselected in accord with ordinary practice. Tablets will containexcipients, glidants, fillers, binders and the like. Aqueousformulations are prepared in sterile form, and when intended fordelivery by other than oral administration generally will be isotonic.Formulations optionally contain excipients such as those set forth inthe “Handbook of Pharmaceutical Excipients” (2005) and include ascorbicacid and other antioxidants, chelating agents such as EDTA,carbohydrates such as dextrin, hydroxyalkylcellulose,hydroxyalkylmethylcellulose and/or organic acids such as oleic acid orstearic add.

The term “pharmaceutically acceptable carrier” as used herein means anymaterial or substance formulated with the active ingredient in order tofacilitate its preparation and/or its application or dissemination tothe site to be treated. Suitable pharmaceutical carriers for use in thecompositions of this invention are well known to those skilled in theart. They include additives such as wetting agents, dispersing agents,adhesives, emulsifying agents, solvents, glidants, coatings,antibacterial and antifungal agents (for example phenol, sorbic acid,chlorobutanol), and isotonic agents (such as sugars or sodium chloride),provided that the same are consistent with pharmaceutical practice, i.e.they are not toxic to mammals.

The pharmaceutical compositions of the present invention are prepared inany known manner, for instance by homogeneously mixing, coating and/orgrinding the active ingredients in a one-step or multi-step procedure,with the selected carrier material and, where appropriate, otheradditives such as surface-active agents. Compositions containing thecompound of this invention formulated into microspheres (usually havinga diameter of about 1 to 10 gm) are useful as controlled or sustainedrelease formulations.

In one optional formulation, compound (1) is comminuted to a finelydivided form, typically to an average particle size at any point withinthe range of about 1-20 microns. The product of example 1 is rods orneedles and exhibits a range of crystal length, typically about 25-40microns. These optionally are micronized in a Jet mill-00 at about 60-80psi to obtain particles of about 3-4 microns and having surface area ofabout 7-8 square meters/g. However, the starting crystal sizes will varyfrom lot to lot and the degree of micronization is a matter of choice.Accordingly, micronized crystalline compound (1) is simply defined ascrystal or amorphous compound (1) that has been subject to amicronization process such as the exemplary one described here. Neitherthe size nor surface area of the resulting particles is critical. Themicronized compound (1) is suspended in aqueous solution, optionallyaided by a suspending agent, emulsifiers and/or surfactant as furtherdescribed below.

Typically, the pharmaceutical formulation is a solubilized form ofcompound (1) where crystalline compound (1) is dissolved in anappropriate solvent or solubilizing agent, or combinations thereof.Crystalline compound (1) is solubilized in a pharmaceutically acceptableexcipient for administration therapeutically or prophylactically.

Suitable solutions of compound (1) for pharmaceutical preparationsinclude water together with various organic acids (typically C4-C24)usually fatty acids like capric, oleic, lauric, capric, palmitic and/ormyristic acid. The fatty acids are optionally saturated or unsaturated,or mixtures thereof. In addition, polyethylene glycols (PEGs) and/orshort, medium, or long chain mono, di, or triglycerides are employedsupplementary to, or in place of, the organic acids. Pegylated short,medium or long chain fatty acids optionally also are used in the samefashion.

The most common organic acids are the carboxylic acids whose acidity isassociated with the carboxyl group —COOH. Sulfonic acids, containing thegroup OSO₃H, are relatively stronger acids for use herein. In general,the acid desirably contains a lipophilic domain. Mono- or di-carboxylicacids are suitable.

Suitable surface-active agents optionally are used with any of theformulations of this invention (any one or more of the following agents,typically any one of them). Such agents also are known as emulgents oremulsifiers, and are useful in the pharmaceutical compositions of thepresent invention. They are non-ionic, cationic and/or anionic materialshaving suitable emulsifying, dispersing and/or wetting properties.Suitable anionic surfactants include both water-soluble soaps andwater-soluble synthetic surface-active agents. Suitable soaps arealkaline or alkaline-earth metal salts, unsubstituted or substitutedammonium salts of higher fatty acids (C₁₀-C₂₂), e.g. the sodium orpotassium salts of oleic or stearic acid, or of natural fatty acidmixtures obtainable from coconut oil or tallow oil. Syntheticsurfactants include sodium or calcium salts of polyacrylic acids; fattysulphonates and sulphates; sulphonated benzimidazole derivatives andalkylarylsulphonates. Fatty sulphonates or sulphates are usually in theform of alkaline or alkaline-earth metal salts, unsubstituted ammoniumsalts or ammonium salts substituted with an alkyl or acyl radical havingfrom 8 to 22 carbon atoms, e.g. the sodium or calcium salt oflignosulphonic acid or dodecylsulphonic acid or a mixture of fattyalcohol sulphates obtained from natural fatty acids, alkaline oralkaline-earth metal salts of sulphuric or sulphonic acid esters (suchas sodium lauryl sulphate) and sulphonic acids of fatty alcohol/ethyleneoxide adducts. Suitable sulphonated benzimidazole derivatives preferablycontain 8 to 22 carbon atoms. Examples of alkylarylsulphonates are thesodium, calcium or alcoholamine salts of dodecylbenzene sulphonic acidor dibutyl-naphthalenesulphonic acid or a naphthalene-sulphonicacid/formaldehyde condensation product. Also suitable are thecorresponding phosphates, e.g. salts of phosphoric acid ester and anadduct of p-nonylphenol with ethylene and/or propylene oxide, orphospholipids. Suitable phospholipids for this purpose are the natural(originating from animal or plant cells) or synthetic phospholipids ofthe cephalin or lecithin type such as e.g. phosphatidylethanolamine,phosphatidylserine, phosphatidylglycerine, lysolecithin, cardiolipin,dioctanylphosphatidyl-choline, dipalmitoylphoshatidyl-choline and theirmixtures. Aqueous emulsions with such agents are within the scope ofthis invention.

Suitable non-ionic surfactants include polyethoxylated andpolypropoxylated derivatives of alkylphenols, fatty alcohols, fattyacids, aliphatic amines or amides containing at least 12 carbon atoms inthe molecule, alkylarenesulphonates and dialkylsulphosuccinates, such aspolyglycol ether derivatives of aliphatic and cycloaliphatic alcohols,saturated and unsaturated fatty acids and alkylphenols, said derivativespreferably containing 3 to 10 glycol ether groups and 8 to 20 carbonatoms in the (aliphatic) hydrocarbon moiety and 6 to 18 carbon atoms inthe alkyl moiety of the alkylphenol. Further suitable non-ionicsurfactants are water-soluble adducts of polyethylene oxide withpoylypropylene glycol, ethylenediaminopolypropylene glycol containing 1to 10 carbon atoms in the alkyl chain, which adducts contain 20 to 250ethyleneglycol ether groups and/or 10 to 100 propyleneglycol ethergroups. Such compounds usually contain from 1 to 5 ethyleneglycol unitsper propyleneglycol unit. Representative examples of non-ionicsurfactants are nonylphenol-polyethoxyethanol, castor oil polyglycolicethers, polypropylene/polyethylene oxide adducts,tributylphenoxypolyethoxyethanol, polyethyleneglycol andoctylphenoxypolyethoxyethanol. Fatty acid esters of polyethylenesorbitan (such as polyoxyethylene sorbitan trioleate), glycerol,sorbitan, sucrose and pentaerythritol are also suitable non-ionicsurfactants.

Suitable cationic surfactants include quaternary ammonium salts,particularly halides, having 4 hydrocarbon radicals optionallysubstituted with halo, phenyl, substituted phenyl or hydroxy; forinstance quaternary ammonium salts containing as N-substituent at leastone C8-C22 alkyl radical (e.g. cetyl, lauryl, palmityl, myristyl andoleyl) and, as further substituents, unsubstituted or halogenated loweralkyl, benzyl and/or hydroxy-lower alkyl radicals.

A more detailed description of surface-active agents suitable for thispurpose is found in “McCutcheon's Detergents and Emulsifiers Annual” (MCPublishing Crop., Ridgewood, N.J., 1981), “Tensid-Taschenbucw”, 2nd ed.(Hanser Verlag, Vienna, 1981) and “Encyclopedia of Surfactants,”(Chemical Publishing Co., New York, 1981).

The compound of this invention is administered by any route appropriateto the condition to be treated, such as oral, rectal, nasal, topical(including ocular, buccal and sublingual), vaginal and parenteral(including subcutaneous, intramuscular, intravenous, intradermal,intrathecal and epidural). The preferred route of administration mayvary with for example the condition of the recipient, but is generallyoral.

Formulations of the compound of this invention for oral administrationusually are presented as discrete units such as capsules, cachets ortablets each containing a predetermined amount of the active ingredient;as a powder or granular form; as a solution or suspension in an aqueousliquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion ora water-in-oil liquid emulsion. The compound of this inventionoptionally is presented as a bolus, electuary or paste.

A tablet is made by compression or molding, optionally with one or moreaccessory ingredients. Compressed tablets are prepared by compressing ina suitable machine the compound of the invention in a free-flowing formsuch as a powder or granules, optionally mixed with a binder, lubricant,inert diluent, preservative, surface active and/or dispersing agent.Molded tablets typically are made by molding in a suitable machine amixture of the powdered compound moistened with an inert liquid diluent.The tablets may optionally be coated or scored and may be formulated soas to provide slow or controlled release of the active ingredienttherein.

The formulations are optionally applied as a topical ointment or creamcontaining the active ingredient(s) in an amount of, for example, 0.075to 20% w/w (including active ingredient(s) in a range between 0.1% and20% in increments of 0.1% w/w such as 0.6% w/w, 0.7% w/w, etc),preferably 0.2 to 15% w/w and most preferably 0.5 to 10% w/w. Whenformulated in an ointment, the compound is employed with a paraffinic ora water-miscible ointment base. Alternatively, the compound isformulated in a cream with an oil-in-water cream base. If desired, theaqueous phase of the cream base may include, for example, at least 30%w/w of a polyhydric alcohol, i.e. an alcohol having two or more hydroxylgroups such as propylene glycol, butane. 1,3-diol, mannitol, sorbitol,glycerol and polyethylene glycol (including PEG400) and mixturesthereof. The topical formulations may desirably include a compound whichenhances absorption or penetration of the active ingredient through theskin or other affected areas. Examples of such dermal penetrationenhancers include dimethylsulfoxide and related analogs.

The oily phase of the emulsions of this invention is constituted fromknown ingredients in a known manner. While this phase may comprisemerely an emulsifier (otherwise known as an emulgent), it desirablycomprises a mixture of at least one emulsifier with a fat or an oil orwith both a fat and an oil. Optionally, a hydrophilic emulsifier isincluded together with a lipophilic emulsifier which acts as astabilizer. It is also preferred to include both an oil and a fat.Together, the emulsifier(s) with or without stabilizer(s) make up theso-called emulsifying wax, and the wax together with the oil and fatmake up the so-called emulsifying ointment base which forms the oilydispersed phase of the cream formulations.

The choice of suitable oils or fats for the formulation is based onachieving the desired cosmetic properties. Thus the cream shouldoptionally be a non-greasy, non-staining and washable product withsuitable consistency to avoid leakage from tubes or other containers.Straight or branched chain, mono- or dibasic alkyl esters such asdi-isoadipate, isocetyl stearate, propylene glycol diester of coconutfatty acids, isopropyl myristate, decyl oleate, isopropyl palmitate,butyl stearate, 2-ethylhexyl palmitate or a blend of branched chainesters known as Crodamol CAP may be used, the last three being preferredesters. These may be used alone or in combination depending on theproperties required. Alternatively, high melting point lipids such aswhite soft paraffin and/or liquid paraffin or other mineral oils can beused.

Formulations suitable for topical administration to the eye also includeeye drops wherein the active ingredient is dissolved or suspended in asuitable carrier, especially an aqueous solvent for the activeingredient. The active ingredient is optionally present in suchformulations in a concentration of 0.5 to 20%, advantageously 0.5 to 10%particularly about 1.5% w/W.

Formulations suitable for topical administration in the mouth includelozenges comprising the active ingredient in a flavored basis, usuallysucrose and acacia or tragacanth; pastilles comprising the activeingredient in an inert basis such as gelatin and glycerin, or sucroseand acacia; and mouthwashes comprising the active ingredient in asuitable liquid carrier.

Formulations for rectal administration may be presented as a suppositorywith a suitable base comprising for example cocoa butter or asalicylate. Formulations suitable for nasal administration wherein thecarrier is a solid include a coarse powder having a particle size forexample in the range 20 to 500 microns (including particle sizes in arange between 20 and 500 microns in increments of 5 microns such as 30microns, 35 microns, etc), which is administered by aerosol or powderinhalers, of which numerous examples are available. Suitableformulations wherein the carrier is a liquid, for administration as forexample a nasal spray or as nasal drops, include aqueous or oilysolutions of the active ingredient.

Formulations suitable for vaginal administration may be presented aspessaries, tampons, creams, gels, pastes, foams or spray formulationscontaining in addition to the active ingredient such carriers as areknown in the art to be appropriate.

Formulations suitable for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain anti-oxidants,buffers, bacteriostats and solutes which render the formulation isotonicwith the blood of the intended recipient; and aqueous and non-aqueoussterile suspensions which may include suspending agents and thickeningagents. The formulations are presented in unit-dose or multi-dosecontainers, for example sealed ampoules and vials, and may be stored ina freeze-dried (lyophilized) condition requiring only the addition ofthe sterile liquid carrier, for example water for injections,immediately prior to use. Extemporaneous injection solutions andsuspensions may be prepared from sterile powders, granules and tabletsof the kind previously described.

The compound of this invention optionally is formulated into controlledrelease compositions in which the release of the compound is controlledand regulated to allow less frequency dosing or to improve thepharmacokinetic or toxicity profile of the invention compound.Controlled release compositions are prepared in accord with knownmethods, many of which involve formulating the active compound with oneor more polymer carriers such a polyester, polyamino acid, polyvinylpyrrolidone, ethylene-vinyl acetate copolymer, methylcellulose,carboxymethylcellulose and/or protamine sulfate. The rate of drugrelease and duration of action optionally is controlled by incorporatingthe active ingredient into particles, e.g. microcapsules, of a polymericsubstance such as hydrogels, polylactic acid, hydroxymethylcellulose,polymethyl methacrylate and the other above-described polymers. Alsosuitable are colloid drug delivery systems such as liposomes,microspheres, microemulsions, nanoparticles, nanocapsules and so on.Depending on the route of administration, the pharmaceuticalcomposition, e.g., tablets, may require protective coatings.

The invention will be more fully appreciated by reference to thefollowing examples, which are to be considered merely illustrative andnot limiting the scope of the invention.

Composition percentages are by weight unless otherwise apparent from thecontext.

EXAMPLE 1 Synthesis of Crystalline5-((6-(2,4-Bis(trifluoromethyl)phenyl]pyridazin-3-yl)methyl)-2-(2-fluorophenyl)-5H-imidazo[4,5-c]pyridine

To a reactor, containing 2,4-bis(trifluoromethyl)bromobenzene (1.00 eq)and tetrahydrofuran (THF), was charged Isopropyl magnesium chloride(¹PrMgCl) (2 M in THF, 1.14 eq) while maintaining the content at −10° C.The mixture was agitated at −10° C. until the reaction was completed byHPLC analysis. The resultant mixture was transferred to the secondreactor, containing trimethyl borate (2.26 eq) and THF held at atemperature of −10° C. The reaction was then monitored by HPLC until1,3-bis(trifluoromethyl)benzene was not more than 2%. Aq. HCl (aqueoushydrochloric acid), prepared from water and concentrated 37%hydrochloric acid (HCl) were then added to quench the reaction whilemaintaining the content at not more than 25° C. After agitating thecontent for 1-2 h and settling for ca. 30 minutes, the layers wereseparated. The organic layer was washed with brine solution mixed withwater and then concentrated under vacuum. Heptane was charged and thecontent was further concentrated under vacuum. The operations wererepeated one more time. Heptane was then charged and the resultantslurry is cooled to 3° C., and agitated at the temperature for 4-6 h.

The product was filtered, washed with heptane twice and dried undervacuum at a maximum of 40° C.

v/w w/w Mole Material M.W. Ratio Ratio Ratio 2,4-Bis(trifluoromethyl)-293.00 — 1.00 1.00 bromobenzene Heptanes 100.21 13.20  9.00 —Hydrochloric acid, 36.50 0.42 0.50 — concentrated (37%) Isopropylmagnesium chloride 102.85 1.95 1.90 1.14 (2 M in Tetrahydrofuran) SodiumChloride (NaCl) 58.11 — 0.60 — Tetrahydrofuran (THF) 72.11 4.50 4.00 —Trimethyl borate 103.91 0.86 0.80 2.26 Water 18.02 8.90 8.90 —

3-Chloro-6-methylpyridazine (1.00 eq), 2-(dicyclohexylphosphino)biphenyl(0.05 eq), 2,4-bis(trifluoromethyl)phenylboronic acid (1.85 eq),1,2-dimethoxyethane and aqueous potassium carbonate solution were allcharged into a reactor. After degassing three times with nitrogen,palladium acetate (0.025 eq) was charged and the content is heated andagitated under reflux until the reaction was deemed complete.

The reaction mixture was cooled to 22° C. Heptane was charged, followedby addition of Celite. After agitating for ca. 30 minutes at 22° C., themixture was filtered into the first reactor, rinsing forward with amixture of 1,2-dimethoxyethane and Heptanes. The layers of the filtrateare separated.

To the organic layer was charged borane trimethylamine complex (0.03eq), water, and acetic acid. The resultant mixture with a pH at maximum4 was agitated for 1-2 h at 22° C. and then refluxed at ca. 80° C. for2-3 h. After cooling back to 22° C., the mixture was adjusted to pH10-11 with addition of 5% aq. sodium hydroxide while maintaining thecontent at 22° C. and then agitated for 1-2 h. The mixture was filteredand the layers were separated. The aq. layer was disposed of and theorganic layer was filtered through ZetaCarbon cartridges into thein-process cleaned first reactor, rinsing forward with1,2-dimethoxyethane through the carbon cartridges.

The filtrate was concentrated under vacuum with a maximum jacket settingof 60° C. Heptane was charged and the contents were further concentratedunder vacuum with a maximum jacket setting of 60° C. Additional Heptanewas charged to the concentrate and the 1,2-dimethoxyethane (DME) content(maximum 0.5%) of the mixture was checked by NMR. After adjusting to 85°C. and agitating for ca. 1 h, the mixture was polished filtered hotthrough a filter into the second reactor.

The filtrate in the second reactor was adjusted to reflux and thenagitated for 1 h. With ramp cooling and moderate agitation, the mixtureis cooled from reflux to 0 to 6° C. over a period of minimum 4 h andthen agitated at 0 to 6° C. for 1 h.

The product was filtered, washed with ambient temperature Heptanes anddried under vacuum at a maximum of 40° C. until loss on drying ismaximum 1%.

w/w Mole v/w Materials M.W. Ratio Ratio Ratio2,4-Bis(trifluoromethyl)phenyl- 257.92 4.00 1.85 — boronic acid Boranetrimethylamine complex 72.92 0.018 0.03 — 3-Chloro-6-methylpyridazine128.56 1.00 1.00 — Diatomaceous earth (celite) N/A 0.30 — —Di(cyclohexyl)phosphinobiphenyl 350.49 0.14 0.05 — 1,2-Dimethoxyethane90.12 12.00 — 13.80 Drinking water 18.02 3.75 —  3.75 Glacial aceticacid 60.05 0.05 0.10 — Heptanes 100.21 20.40 — 29.80 Palladium (II)acetate 224.49 0.044  0.025 — Potassium carbonate, 138.21 2.15 2.00 —Sodium hydroxide, 5% solution 40.00 — — —

To a reactor was charged methanesulfonic acid, followed by phosphoruspentoxide (1.00 eq) in portions while maintaining the content at 23° C.3,4-Diaminopyridine (1.00 eq) was charged in portions while maintainingthe content at 20 to a maximum of 50° C. 2-Fluorobenzoic acid (1.09 eq)was then charged. The mixture was heated to 100° C. and the reaction wasmonitored by HPLC until completion.

The content was adjusted to 10° C. and water was charged whilemaintaining the content at a maximum of 25° C. After agitating themixture at this temperature for 1 h, it was filtered into a secondreactor.

To the filtrate in the second reactor was charged 27% ammonium hydroxideuntil the pH was in between 6.0-6.5. The content temperature was kept ata maximum of 30° C. The resultant thin slurry was agitated at 22° C. fora minimum of 1 h and 27% ammonium hydroxide was further charged, untilthe pH was between 8.0-9.3. The slurry was further agitated at 22° C.for a minimum of 2 h.

The product was filtered, washed with water twice, and dried at amaximum of 60° C. under vacuum, until the water content is not more than1%. If necessary, the product is milled to remove large lumps.

w/w Mole v/w Materials M.W. Ratio Ratio Ratio Ammonium hydroxide, 27%35.05 — — — 3,4-Diaminopyridine 109.13 1.00 1.00 — Drinking water 18.0224.00 — 24.00 2-Fluorobenzoic acid 140.11 1.40 1.09 — Methanesulfonicacid 96.10 7.00 —  4.70 Phosphorous pentoxide 141.94 1.30 1.00 —

To a reactor is charged compound 2a (1.24 eq), methylene chloride andtrichloroisocyanuric acid (0.491 eq). The mixture wxas adjusted toreflux and agitated under reflux until the reaction is complete.

The reaction mixture was cooled to 22° C. and celite was charged. Afteragitating for minimum of 30 minutes, the mixture was filtered into andrinsed forward with methylene chloride 3 times into the second reactor.The filter cake was disposed of. To the filtrate in the second reactorwas charged 3% aq. sodium hydroxide whilst maintaining the contents at22° C. The mixture was agitated for 1-2 h and the layers were separated.The bottom organic layer was transferred to the in-process cleaned firstreactor and concentrated under vacuum with a maximum jacket temperatureof 45° C. Methylene chloride was charged and the mixture was polishfiltered to the in-process cleaned second reactor.

The filtrate was concentrated under vacuum with a maximum jackettemperature of 45° C. Dimethylformamide (DMF) was charged and thecontents are further concentrated. The mixture was adjusted to 22° C.and DMF was charged, followed by compound core 2 (1.00 eq) and 10% aq.sodium hydroxide while maintaining the content at 22° C. The resultantmixture was agitated at 22° C. until the reaction was monitored by HPLCanalysis. Over the reaction period, the pH of the content was monitoredand 10% aq. sodium hydroxide was added as required to maintain the pH at11-12 by pH meter. After the reaction, 10% aq. sodium hydroxide wascharged while maintaining the contents at 22° C. The mixture was dilutedwith DMF and agitated for 2 h. The mixture was filtered over a minimumof 1 h into the first in-process cleaned first reactor, containingwater, whilst maintaining the contents at 16° C. and then rinsingforward with DMF. The resultant slurry was agitated for 1-3 h at 22° C.

The crude product was filtered and washed with water and then methyltertiary butyl ether (MTBE). The wet crude product was discharged fromthe filter and transferred into the first reactor; and ethyl acetate(EtOAc) was charged. The mixture was heated to reflux and agitated atreflux temperature until all the solids are dissolved. The water levelmust be less than 6.0%. With ramp cooling, the content was adjusted to22° C. over a minimum of 4 h.

The crystallized product was filtered and washed with EtOAc and thencharged back to the first reactor. Ethyl acetate (EtOAc) was added. Themixture was heated to reflux and agitated at the temperature until allthe solids are dissolved. The water level must be not less than 1.0%.The mixture was filtered, hot, through a polishing filter into thesecond reactor (EtOAC preconditioned), rinsed forward with EtOAc.

The product was concentrated under atmospheric pressure. After adjustingto 65° C. and charging in EtOAc, the pot was adjusted to reflux andagitated at reflux for ca. 30 minutes. Water content was checked and ifthe water level was more than 0.2%, the same cycle was repeated.

Once the water level was at maximum 0.2%, the content was adjusted toreflux and then agitated under reflux for 1-3 h. With ramp cooling, thecontent was adjusted to 22° C. over minimum 4 h and then agitated at thetemperature for minimum of 8 h.

The product was filtered, washed with EtOAc and dried under vacuum atmaximum of 60° C. The product was then milled.

w/w Mole v/w Materials M.W. Ratio Ratio Ratio3-(2,4-Bis(trifluoromethyl)- 306.21 1.00 1.00 —phenyl)-6-methylpyridazine, t-Butyl methyl ether 88.15 — — —Diatomaceous Earth (Celite) — — N,N-Dimethylformamide 73.10 6.90 —  7.30Drinking water 18.02 27.72 — 27.72 Ethyl acetate 88.11 33.90 — 37.702-(2-Fluorophenyl)-imidazo-[4,5- 213.21 0.560 0.78 — c]pyridine, GS-9133Methylene chloride 84.93 16.50 — 12.50 Sodium hydroxide 40.00 0.276 — —Trichloroisocyanuric acid 232.41 0.315  0.415 —

Nuclear Magnetic Resonance (¹H-, ¹³C-, and ¹⁹F-NMR) Spectra

Nuclear magnetic resonance (NMR) spectra of compound (1) is consistentwith the proposed structure. The ¹³C, ¹⁹F, and ¹H-NMR spectra ofcompound (1) in DMSO-d₆ were measured using a Varian UnityInova-400FT-NMR spectrometer. Spectra are shown in the table below. The NMRchemical shift assignments were established using 2D correlationexperiments (COSY, HSQC, HMBC and HSQCTOCSY).

¹H- and ¹³C-NMR Chemical Shift Assignments for Compound (1) ReferenceStandard

δF/ppm (DMSO- Atom δC/ppm (DMSO-d₆) d₆) δH/ppm (DMSO-d₆) 1A 140.16 2A128.32 (q^(a), JCF = 32 Hz) 3A 123.61, m 8.24 (m, 1H) 4A 130.27 (q, JCF= 34 Hz) 5A 129.54 (q, JCF = 3 Hz) 8.22 (m, 1H) 6A 133.36 7.88 (m, 1H)7A 123.20 (q, JCF = 273 Hz) −56.4^(b) 8A 123.02 (q, JCF = 275 Hz)−62.0^(b) 1 158.76 2B 128.16 8.01 (d, 1H, J = 8.4 Hz) 3B 126.20 7.95 (d,1H, J = 8.8 Hz) 4B 157.70 5B  60.49 6.17 (s, 2H) 2C 131.86 8.31 (m, 1H)3C 112.63 7.86 (m, 1H) 4C 155.44 6C 168.11 (d, JCF = 6 Hz) 8C 145.08 9C133.06 9.25 (s, 1H) 1D 123.11 (d, JCF = 10 Hz) 2D 160.46 (d, JCF = 254Hz) −111.7 3D 116.59 (d, JCF = 22 Hz) 7.29 (m, 1H) 4D 130.84 (d, JCF = 8Hz) 7.46 (m, 1H) 5D 124.13 (d, JCF = 4 Hz) 7.31 (m, 1H) 6D 131.72 (d,JCF = 2 Hz) 8.35 (m, 1H) ^(a)multiplicity, s: singlet, d: doublet, q:quartet, m: multiplet ^(b)interchangeable signals

Differential Scanning Calorimetry

Compound (1) samples (amorphous) designated “Research lot 6” were madeaccording to the method published as Example 1a in WO 08/005,519, whichis hereby incorporated by reference in its entirety. The remainingsamples were crystalline compound (1). The samples were subjected tomeasurement using a Differential Scanning Calorimetry (DSC) apparatus(DSC2010, manufactured by TA Instruments Corporation), under nitrogenatmosphere, sample weight 5±1 mg, temperature rise rate: either 1° C.per min, 5° C. per min or 10° C. per min, open aluminum pan, and indiumstandard as a reference. The enthalpy, extrapolated onset temperatureand apex temperature at an endothermic peak on the obtained DSC curvewere determined.

The DSC results for Research lot 6 and representative crystalline freebase compound (1) batches are summarized in Table 1 and FIGS. 4 and 5,respectively. When the crystal form of compound (1) was subjected to DSCscan at 1° C./min, the enthalpy of the endothermic peak is about 80 J/g,and the extrapolated onset temperature is 233.2° C.±2.0° C. The apex ofthe endothermic peak is 233.9° C.±3.0° C.

TABLE 1 Example DSC values obtained for Compound (1) batches 10° C./ minscan 1° C./min peak scan Enthalpy onset main peak peak onset main peak(J/g) 9190 Ref Std 235.8 237.2 233.7 234.6 89.5 9190-A-1 n/a n/a 234.8234.0 — 9190-B-1 Crop 1 235.2 237.4 231.6 232.2 78.5 9190-B-1 Crop 2236.1 238.5 234.3 235.6 80.9 **Research Lot 6 220.2 221.3 pendingpending 39.1 Note: All ° C. excecpt for enthalpy **5° C./min scanreported for Lot 6

X-Ray Powder Diffractometry Study 1

Samples made by example 1a of WO 05/063744 and by the method of thisinvention were analyzed in the as received condition, only mixing with aspatula prior to analysis. A sample was fixed to an aluminum cell, andthe measurement was performed using an X-ray powder diffractometer(XRD-6000, Shimadzu Lab X, manufactured by Shimadzu Corporation, X-raysource: Cu—Kα1 ray, tube voltage: 35 kV, tube electric current: 40 mA,scan speed: 2° per min, continuous scan mode, sampling pitch: 0.02°,scan range: 4-35°, β axis rotation: 60 rpm).

Non-micronized, ascicular compound (1) crystals obtained by the methodof this invention have an X-ray powder diffraction pattern havingcharacteristic diffraction peaks at diffraction angles 2θ (°) of 13.46,15.59, 16.90, 17.48, 23.05 and 30.15 as measured by X-ray powderdiffractometer (FIG. 1). Note that the non-micronized “high melt” 235°C. melt ascicular crystal form of compound (1) tested in this exampleshows some effects due to preferred orientation and particle size. As aresult, FIG. 1 should be considered merely exemplary because varying thecrystal size and orientation will change the magnitude of the peaks inthe plot. Additionally, the diffraction peak value at the abovementioned diffraction angle 2θ (°) may show slight measurement error dueto the measurement instrument or measurement conditions and the like.Typically, the measurement error generally is within the range of about±0.3. The specification for the Shimadzu XRD-6000 is ±0.04. Further,some variation in peak positions can be expected due to product andexperimental variation, so they must be considered approximate.

The 220° C. “low melt” solid state form of compound (1) comprised byproduct made according to the example 1a method (or in the method hereinprior to the reslurry step) gives an X-ray powder diffraction patternconsistent with amorphous material (FIG. 3).

Compound (1) by the method of this invention typically exhibitsintrinsic solubility of 0.7 micrograms/ml, a pKa of 5.8, log P of 2.8;and geometric mean (3 lots) pH solubility profile at pH 2 of 458micrograms/ml and at pH 7.3, 0.7 micrograms/ml. Geometric meansolubility (3 lots) in simulated intestinal fluids (fasted: pH 6.4, 0.75mM lecithin, 3 mM sodium taurocholate, 270 mOsmol; fed: pH 5.0, 3.75 mMlecithin, 15 mM sodium taurocholate, 635 mOsmol) were 19.1 micrograms/ml(fasted) and 122 micrograms/ml (fed).

Measured parameters vary from lot to lot, so all of the foregoingparameters except molecular weight should be considered to beapproximate.

Titration with acids revealed higher solubility with mesylate (>20mg/ml) compared to the chloride (about 0.6 mg/mL) or sulfate (about 0.5mg/mL) counterions.

X-Ray Powder Diffractometry Study 2

Another sample of crystalline compound (1) prepared by the method ofthis invention was analyzed in the same fashion as Study 1 except thatthe X-ray powder diffractometer was a PANalytical X'Pert Pro MPD PW3040Pro, manufactured by PANalytical Inc., using X-ray source: Cu—Kα ray(1.54059 Å), tube voltage: 45 kV, amperage: 40 mA, scan range: 1-55° 20,step size: 0.008° 2θ, collection time: 3373 s, scan speed: 0.9° per min,slit: DS: ½°, SS: ¼°, revolution time: 0.5 s, mode: transmission. Theresults are depicted in FIG. 2.

EXAMPLE 2 Formulation of Compositions Using Compound (1)

Crystalline compound (1) is used as an intermediate to producepharmaceutically acceptable solutions. The following examples are madeon a weight by weight basis to achieve 10% w/w active. To make 12 kgsolution, exemplary quantitative compositions of compound (1) capsules,20 mg and 40 mg are listed below.

Quantitative composition of Compound (1) capsules, 20 mg and 40 mg

Capsule Unit Formula (mg/unit) Compendial Components % w/w 20 mg 40 mgReference Function Compound 1 10.00 20.0 40.0 None Active ingredientOleic Acid 84.55 169.1 338.2 NF Solvent Polysorbate 80 5.00 10.0 20.0 NFSurfactant Butylated Hydroxytoluene 0.10 0.2 0.4 NF Antioxidant (BHT)Butylated Hydroxyanisole 0.35 0.7 1.4 NF Antioxidant (BHA) CapsuleSealing Solution^(a) Capsule sealant Ethanol —^(b) —^(b) —^(b) USP —Purified water —^(b) —^(b) —^(b) USP — Capsule Shell, Size 0 Licaps ™N/A 1 each 1 each None Capsule shell White Opaque Total 100.00 200.0400.0 ^(a)Composition is 1:1 w/w ethanol:water solution. ^(b)Removedduring the capsule sealing process.

Container/vessel: 12 kg stainless steel

Weigh the following in order:

0.012 kg butylated hydroxytoluene (0.10%)

0.035 kg butylated hydroxyanisole (0.35%)

1.2 kg Compound (1) free base (10%).

0.6 kg Polysorbate 80 (5%) weighed

10.153 kg oleic Acid (equivalent to 84.55 g (84.55%))

Solubilized crystalline compound (1) capsules, 20 mg or 40 mg, aremanufactured through a series of unit process steps. Compound (1) drugsubstance, oleic acid, polysorbate 80, butylated hydroxytoluene (BHT),and butylated hydroxyanisole (BHA) are mixed until a solution isachieved. The solution is filled into 2-piece hard gelatin capsules.Closed capsules are subsequently sealed with a hydroalcoholic solution,which is evaporated during the sealing process. A vacuum leak test isperformed on sealed capsules prior to packaging.

Alternative Formulations

The crystalline compound of formula (1) optionally is used as anintermediate to be formulated into a solubilized form with the followingagents:

-   -   Fatty acids (short, medium, and long chained as well as        saturated and unsaturated), typically C4 to C22. Typical fatty        adds are linoleic acid, lauric acid, capric acid or oleic acid.    -   Alcohols such as ethanol, benzyl alcohol, glycerol, polyethylene        glycol 200, polyethylene glycol 300, polyethylene glycol 400.    -   Surfactants, including both ionic and non-ionic surfactants.        Examples of non-ionic surfactants are fatty acid esters of        polyoxyethylene sorbitan, sorbitan fatty acid ester,        polyoxyethylene castor oil derivatives, polyoxyethleneglycerol        oxystearate, polyethyleneglycol 60, hydrogenated castor oil,        and/or block copolymers of ethylene oxide and propylene oxide.    -   Antioxidants, for example butylated hydroxyanisole (BHA),        butylated hydroxytoluene (BHT), ascorbyl palmitate, vitamin E,        and/or vitamin E PEG 1000 succinate for chemical stability.    -   Viscosity inducer (silicon dioxide, polyethylene glycols,        titanium oxide and the like).    -   And mixtures of the above

Encapsulation can be performed in a soft elastic gelatin or a hardgelatin or a hard hydroxypropyl methyl cellulose capsule. The liquidformulation (solution or encapsulated solution) provides improved oralbioavailability.

Capsule Filling

The composition and preparation of the soft elastic gelatin capsule iswell known in the art. The composition typically comprises from 30-50%by weight gelatin, 10-40% plasticizer or a blend of plasticizers andabout 25-40% by weight water. Plasticizers can be glycerin, sorbitol orsorbitol derivatives, propylene glycol and the like or a combinationthereof.

Various methods can be used for manufacturing and filling the softelastic gelatin capsules such as rotary, liner or accogel machine andthe like. Hard gelatin or HPMC capsules can be purchased from Capsugel,Greenwood, S.C. and other suppliers. Capsules are filled manually or bycapsule filling machine.

Formulation Preparation

In general, the compositions of this invention can be prepared in thefollowing manner. The ingredients are mixed in an appropriate vesselsize using an overhead mixer (The mixing tank may be purged withnitrogen). The pharmaceutically acceptable fatty acid and thepharmaceutically acceptable antioxidant are mixed at room temperature.(The solution may be warmed to appropriate temperature if needed, forexample to about 45° C. in the case of lauric acid, in order to liquefythe fatty acid). The compound of formula (1) is added and stirred untildissolved. The pharmaceutically acceptable surfactant is added withmixing. The appropriate weight of the resulting mixture is filled intohard gelatin capsules

Additional Formulation Compositions

Formula (1) 8.0 compound PEG 400 82.8 EtOH 9.2 Total 100.0

Formula (1) 8.0 compound EtOH 11.0 PG 7.4 Maisine 35-1 36.8 Cremophor36.8 RH40 Total 100.0

Formula (1) 8.0 compound Oleic Acid 92.0 Total 100.0

Formula (1) 8.0 compound Oleic Acid 73.6 EtOH 9.2 Tween 20 9.2 Total100.0

Formula (1) 8.00% compound Oleic Acid 87.40% Tween 80 4.60% Total100.00%

FORMULA (1) 20.00% COMPOUND Oleic Acid  80.0% Total 100.0%

FORMULA (1) 20.00% COMPOUND Oleic Acid 76.00% Tween 80 4.00% Total100.00%

FORMULA (1) 8.00 COMPOUND Oleic Acid 86.47%  Tween 80 4.60% Aerosil 2000.92% BHT 0.01% Total 100.0% 

FORMULA (1) 8.00 COMPOUND Oleic Acid 85.55%  Tween 80 4.60% Aerosil 2001.84% BHT 0.01% Total 100.0% 

FORMULA (1) 8.00 COMPOUND Oleic Acid 85.55%  Tween 80 4.60% Aerosil 2001.84% BHT 0.01% Total 100.0% 

FORMULA (1) 10.00 COMPOUND Oleic Acid 84.55% Tween 80  5.00% BHA  0.35%BHT  0.1% Total 100.0%

EXAMPLE 2a Micronized Formulation of Compound (1)

Micronized drug substance (Jet mill-00 at 60-80 psi; 3-4 microns averagesize, about 7-8 sq. meters/g) was dry blended with lactose,microcrystalline cellulose, croscarmellose sodium, sodium laurylsulfate, tartaric acid, and hydroxypropyl cellulose. The blend wasgranulated by spraying the blend solution. The granules were dried in afluid-bed. The dried granules were sized by passing through a mill, andthen blended with additional microcrystalline cellulose andcroscarmellose sodium. The powder blend was lubricated by addingmagnesium stearate and then compressed into tablets using a rotarytablet press. The tablets were subsequently film-coated.

The table below is a summary of various formulations tested in dogsdosed at 40 mg compound (1), corresponding to approximately 4 mg/kg. Thetable illustrates the superior performance of the solubilized compound(1) formulations.

In-vivo Data Summary Drug Load Cmax AUC₂₄ Dosage Form Process Formula(%) (μM) (μM hr) F (%) RSD (%) Solid Powder PIC 50 0.7 2.9 8 52 Fill^(a)Solubilized Liquid Fill Capric acid 20 4.8 25 79 17 Lauric acid 20 2.614.3 44 29 Oleic Acid 8 3.8 23 67 27 20 2.1 14 44 56 25 7.9 42 125 24Solid High SLS only 20 0.4 4.4 13 85 Shear^(a) SLS & 20 0.4 2.7 8 82Tartaric SLS & 20 0.9 6.9 20 67 Tartaric^(b) Fluid SLS & 20 0.3 4.4 1477 bed^(a) Tartaric ^(a)Utilizes micronized API ^(b)Dosed in dogstreated with pentagastrin to reduce stomach pH

EXAMPLE 3 Antiviral Activity of Compound (1)

The compound of this invention exhibits anti-HCV replicon activity(assay described in WO 05/063744) against both genotypes 1a and 1,extremely low cytotoxicity (>50,000 nM in Huh-7, HepG2 and MT4 cells),and a highly favorable selectivity index. The compound is substantiallyless active against genotype 2a.

Activity of Compound 1 Against HCV Genotype 1 and 1a Replicons

HCV genotype 1 (Con-1/lucneo) and 1a (H77/neo) replicon cells wereincubated with serial dilutions of compound (1) 2′C-methyl adenosine(2′CMeA) or IFNα for 3 days in the absence or presence of 40 mg/mL humanserum albumin (HSA). After incubation, replicon RNA levels in thetreated cells were determined by either a luciferase reporter assay (1replicon) or a quantitative real-time PCR assay (1a replicon) and thedata points were used to calculate EC₅₀ (50% effective inhibitingconcentration) values for the inhibitors. Compound (1) was shown toinhibit both genotype 1 and genotype 1a replicons with EC₅₀ values of0.6 and 3.6 nM, respectively (Table A). In the presence of human serumalbumin, the EC₅₀ value of Compound (1) was increased to 11 nM.

TABLE A Activity of Compound (1) against HCV Genotypes 1a and 1Replicons EC₅₀ [nM]^(a) HCV 1-lucneo 40 mg/mL Compound HCV 1-lucneo HSAHCV-1a 1 0.6 ± 0.28  11 3.6 ± 1.4 2′CMeA 175 ± 70  250 170 IFN-α 2 IU/mLn.d. n.d. n.d., not determined; HSA, human serum albumin ^(a)Mean EC₅₀value and standard error determined from at least 4 independentexperiments

Activity of Compound (1) Against HCV Genotype 1a Replicon and Virus

The antiviral activity of compound (1) against HCV genotype 2a wastested in cells chronically infected with the genotype 2a virus as wellas in cells replicating a subgenomic 2a replicon. Huh-7 cells containingchronically replicating HCV genotype 2a (J6/JFH-Rluc) virus orsubgenomic replicons were cultured with compound (1) or 2′CMeA for 3days in the absence of human serum albumin. After cultivation, theamount of luciferase in 2a-virus containing cells and HCV NS3 proteaseactivity in the 2a replicon-containing cells was determined usingPromega's luciferase assay and a novel time—resolved fluorescence assay,respectively.

The antiviral activity of compound (1) was significantly reduced in boththe HCV-2a chronically infected cell culture model (EC₅₀=2.9 μM) and the2a subgenomic replicon model (EC₅₀=21.9 mM) compared to Huh-7 cellsreplicating an HCV-1 subgenomic replicon (EC₅₀=0.0006 mM) (Table 2).Taken together, these results suggest that the reduction in potency forcompound (1) against HCV genotype 2a may be due to the genotypicdifferences between genotype 1 and genotype 2 of HCV.

TABLE B Activity of Compound (1) against HCV Genotypes 1 and 2a EC₅₀[nM]^(a) HCV 1-lucneo HCV 2a (subgenomic (subgenomic HCV-2a Compoundreplicon) replicon) (reporter virus) 1 0.6 ± 0.28 21898 ± 18972 2900 ±1250 2′CMeA 175 ± 70  1610 ± 1099 194 ± 26  IFN-α 2 IU/mL n.d. 1.2 IU/mLn.d., not determined; HSA, human serum albumin ^(a)Mean EC₅₀ value andstandard error determined from at least 4 independent experiments

Compound (1) was evaluated for its cytotoxicity in a variety of celltypes including HCV replicon-containing cell lines (Huh-7, SL3 and MH4)and non-replicon-containing cell lines (HepG2, MT4), using aCellTiter-Glo Luminescence Cell Viability assay (Promega). No toxiceffects were observed in any of the cell lines at the highestconcentration tested (50 μM) (Table C). These results, coupled with itspotent antiviral activity (EC₅₀=0.62-3.6 nM) in HCV-1 and HCV-1areplicons, indicates a high selectivity index(CC₅₀/EC_(50>13,000)-80,000) for compound (1).

TABLE C Cytotoxicity of compound (1) in HCV Replicon Containing CellLines CC₅₀ [μM]^(a) Huh-7 Compound lucneo^(b) SL3^(b) MH4^(b) HepG2 MT41 >50 >50 >50 >50 >50 2′CMeA 7.2 ± 6 3.9 16 24.3 ± 2.1 3.5 ± 1.9 n.d.,not determined; HSA, human serum albumin ^(a)Mean CC₅₀ value andstandard error determined from at least 4 independent experiments^(b)HCV replicon-containing cell lines

Anti-HCV Activity of Compound (1) in Combination with IFN In Vitro

Pegylated interteron-α (PEG-IFN-α), in combination with ribavirin,represents the current standard of care for HCV-infected patients. Invitro combination studies of compound (1) and IFN-α were performed inreplicon cells. Data was analyzed using the MacSynergy templatedeveloped by Prichard and Shipman. Results from these studies suggest anadditive interaction between compound (1) and IFN-α.

EXAMPLE 4 Antiviral, Pharmacokinetic and Safety Data for Compound (1) ina Phase-1, First-in-Human Trial in HCV Genotype 1-Infected Subjects

A randomized, double-blind, placebo controlled trial was designed toevaluate the safety/tolerability, pharmacokinetics and antiviralactivity of single (in Part A) and multiple (in Part B) doses ofCompound (1) (oleic acid solution, above) in subjects chronicallyinfected with HCV genotype 1 (GT-1) without decompensated cirrhosis.Prospective subjects are 18-60 years of age, are HCV treatment naïve,and are in general good health.

In completed Part A, five successive cohorts of 6 subjects wererandomized (5:1) to receive single ascending doses of Compound 1 (40,120, 240, 240—with food, or 480 mg) or placebo. In ongoing Part B, foursuccessive cohorts of 12 subjects are randomized (10:2) to receivemultiple ascending doses of Compound 1 (40 mg BID, 120 mg BID, 240 mgQD, 240 mg BID) or placebo, over 8 days.

Thirty-one subjects enrolled in Part A were of mean age 43.6 years,predominantly male (20/31), Caucasian (25/31), and infected with eitherHCV Genotype-1a (24) or 1 (6). Median (range) baseline HCV viral loadwas 6.6 Log¹⁰ RNA IU/mL (5.2-7.3). Single doses of compound (1) werewell tolerated, with no serious or treatment-limiting adverse events(AEs) reported. The most common AE was headache. All AEs were mild inseverity, with the exception of one moderate headache. There were noGrade 3 or 4 treatment emergent laboratory abnormalities.

Median compound (1) plasma half-life ranged from 10 to 15 hours acrosscohorts. Systemic exposure was increased approximately 2-fold whencompound (1) was administered with a high fat meal. Mean compound (1)concentration 24 hours after the 240 mg fasted dose dosing was ˜7-foldhigher than the protein binding adjusted in vitro HCV GT-1 Replicon EC₅₀value. Following single-dose exposure, maximal antiviral effect wasobserved at 24 hours, with median declines ranging from 0.46 to 1.49Log¹⁰ HCV RNA IU/mL across cohorts. Individual HCV RNA declines amongall compound (1) recipients ranged from 0.19 to 2.54 log¹⁰ IU/mLfollowing single-dose exposure.

This is the first clinical demonstration of antiviral activity ofcompound (1). Single dose exposure to compound (1) was well tolerated,demonstrated favorable PK properties and potent antiviral activity.

1. A crystalline compound of formula (1)

and its salts, which is substantially free of amorphous compound (1). 2.The crystalline compound of claim 1 having an endothermic onset at about234° C. in differential scanning calorimetry (DSC) profile.
 3. Thecrystalline compound of claim 2 having a heat of fusion (DH_(f)) ofabout 89 J/g (42 KJ/mole).
 4. The crystalline compound of claim 3 havingat least one approximate peak at diffraction angle 2θ (°) of about 17 asmeasured by X-ray powder diffractometry.
 5. The crystalline compound ofclaim 1 which is the free base.
 6. The crystalline compound of claim 1in the form of needles or rods.
 7. A composition comprising thecrystalline compound of claim 1 containing less than about 40% by weightof the amorphous polymorph of compound (1).
 8. A composition comprisingthe crystalline compound of claim 7 which contains less than about 10%by weight of amorphous compound (1).
 9. A composition comprising thecrystalline compound of claim 8 wherein the crystalline compound (1)contains less than about 100 ppm chloride.
 10. The crystalline compoundof claim 1 which has been micronized.
 11. The crystalline compound whichis the free base substantially free of amorphous compound (1) and anyother crystal form of compound (1).
 12. The crystalline compound ofclaim 1 which is substantially free of the chloride salt of compound(1).
 13. A composition comprising the crystalline compound of claim 1and a pharmaceutically acceptable excipient.
 14. A method for makingcrystalline compound (1)

comprising crystallizing compound (1) from crystallization solvent andcontrolling the amount of water in the crystallization solvent.
 15. Themethod of claim 14 wherein the compound (1) is crystallized from ethylacetate or ethyl acetate/isopropyl alcohol solvent containing less thanabout 0.9% by weight of water in the solvent.
 16. The method of claim 14wherein the amount of water is controlled such that less than about 10%by weight of amorphous compound (1) is precipitated duringcrystallization.
 17. The method of claim 14 wherein the water iscontrolled by removing it azeotropically from the crystallizationsolvent.
 18. The method of claim 14 comprising providing water in thecrystallization solvent at a concentration of less than about 10%. 19.The method of claim 18 comprising a plurality of crystallization steps,wherein crystallization is effected from solvents comprisingsuccessively lower concentrations of water.
 20. The method of claim 18wherein the last crystallization step is effected from solventcomprising less than about 0.9% water.